Diabetes mellitus is one of the major world health problems. Reduced pancreatic ?-cell function and loss of ?-cell mass are key events in the pathogenesis of diabetes mellitus. In normal conditions, the pancreatic ?-cell responds to elevated blood glucose with insulin secretion, and to persistently elevated blood glucose (hyperglycemia) with compensatory increase in insulin secretion and ?-cell mass. Persistent hyperglycemia may also lead to glucotoxic ?-cell dysfunction, with loss of insulin content and ?-cell mass. We and others have recently demonstrated that loss of ?-cell mass in some forms of diabetes is associated with loss of ?-cell identity, rather than cell death, as frequently assumed. Strikingly, we have also shown that this process is reversible, with recovery of ?-cell mass and therefore antidiabetic drug responsivity, after lowering of blood glucose with insulin therapy in experimental neonatal diabetes mellitus. These results, correlating with human studies of recovery of sulfonylurea responsivity following insulin therapy in diabetes, challenge the current understanding of permanent ?-cell damage in diabetes, and prompt us to study the underlying mechanisms of loss- and recovery- of ?-cell identity in vivo, which remain elusive. Chronic hyperglycemia will lead to hyperstimulated metabolism and increased reactive oxygen species (ROS) formation. Excessive ROS will lead to mitochondrial dysfunction, and increased oxidative and endoplasmic reticulum (ER) stress; likely to be major contributors to glucotoxic loss of ?-cell identity in systemic diabetes. The main goal of this proposal seeks to address the question of what are the mechanisms underlying loss of ?-cell mass and identity in diabetes in vivo. We will use novel mouse models of human neonatal diabetes driven by ?-cell insulin secretory deficiency as well as other diabetic mouse models. We will specifically test what is the contribution of hyperglycemia vs. lack of insulin in loss of ?-cell identity in systemic diabetes, and if hyperglycemia-induced hypermetabolism and consequent mitochondrial dysfunction is driving it. We will also determine translatability of these features to human ?-cells by using stem cell derived ?-cells from diabetic patients. In seeking answers to these questions, the experiments proposed in this project represent a significant effort to understand mechanisms underlying diabetic glucotoxicity and loss of ?-cell identity will be of direct relevance to the progression of human diabetes, potentially opening up the exciting possibility to restore or revent loss of ?-cell mass in diabetes.